PROJECT SUMMARY Neonatal pulmonary hypertension (PH) is a serious condition that affects up to one third of premature infants with bronchopulmonary dysplasia (BPD). This entity, known as BPD-associated PH (BPD-PH), is fully established by 1-3 months of age and incurs a four-fold increased risk of death. Survivors of BPD-PH have prolonged and recurrent hospitalizations, and are at risk for chronic cardiopulmonary and metabolic problems. We are committed to developing targeted strategies to prevent BPD-PH in premature infants. Two promising therapeutic targets are the histologic marker, placental maternal vascular underperfusion (MVU) and the intermediate cord blood-derived fetal monocytes (iMNC) that circulate at birth. Our central hypothesis is that fetal iMNCs are adversely programmed by the chronic fetal hypoxia of placental MVU, and contribute to early delayed lung angiogenesis leading to BPD-PH. In Aim #1, we will elucidate the mechanism by which MVU leads to delayed neonatal angiogenesis. Using fetal growth restriction (FGR, birth weight <5th percentile) as the proxy for chronic fetal hypoxia, we will compare iMNC VEGFR1 expression via flow cytometric analysis of 100 cord blood samples from the following groups of infants: 1) preterm MVU with FGR; 2) preterm MVU without FGR; 3) preterm no MVU with FGR; 4) preterm no MVU no FGR (preterm control); 5) term no MVU no FGR (term control). In isolated iMNCs, we will use PCR Array to compare the gene expression profiles of these groups and identify novel gene targets and pathways driven by MVU. In Aim #2 we will identify the perinatal mechanisms by which MVU-exposed fetal iMNCs contribute to delayed neonatal lung angiogenesis: In cultured iMNCs exposed to experimental hypoxia and hyperoxia, we will evaluate VEGFA and VEGFR1 gene and protein expression. We will perform chemotaxis assays to compare iMNC migration in response to exogenous treatment with placental growth factor (PIGF) and granulocyte colony stimulating factor (GCSF), and we will compare iMNC expression of VEGFR1 in bronchoalveolar lavage fluids from intubated preterm infants. Lastly, in Aim #3 we will transplant iMNCs from the 5 patient groups into newborn human GM-CSF knock-in (humanized) mice, and follow the lung histology and PH parameters after chronic hyperoxia exposure (85% oxygen x 14 days). We will test the hypothesis that transplantation of cord blood-derived iMNCs from non-MVU, non-FGR infants (controls) will aid in the prevention of BPD-PH.